The rapid and accurate detection of target molecules is critical for many areas of research and medical diagnosis. Important features for a diagnostic technique to be used for the detection of analytes are specificity, speed, and sensitivity. Time constraints and ease of on-site analysis can be major limitations.
Most assay systems can be characterized as having three key components: a probe that recognizes the target analyte(s) with a high degree of specificity; a reporter that provides a signal that is qualitatively or quantitatively related to the presence of the target analyte; and a detection system capable of relaying information from the reporter to a mode of interpretation. The probe (e.g., antibody, nucleic acid sequence, or enzyme product/activity) should interact uniquely and with high affinity to the target analyte(s), but not with non-targets. In order to minimize false positive responses, it should not react with non-targets.
The label is often directly or indirectly coupled (conjugated) to the probe, providing a signal that is related to the concentration of analyte upon completion of the assay. The label should not be subject to signal interference from the surrounding matrix, either in the form of signal loss from extinction or by competition from non-specific signal (noise) from other materials in the system.
The detector is usually a device or instrument used to determine the presence of the reporter (and therefore analyte) in the sample. Ideally, the detector should provide an accurate and precise quantitative scale for the measurement of the analyte. In rapid on-site tests, such as pregnancy tests, the detection instrument is the human eye and the test results are qualitative (positive or negative).
Proteases are implicated in disparate pathologies including: virulence factors that facilitate infectious diseases (Matayoshi, E. D. et al. Science, 247 (February 1990): 954-958; Sham, H. L. et al. Journal of Medicinal Chemistry, 39, no. 2 (1996): 392-397; Sham, H. L. et al. Antimicrobial Agents and Chemotherapy, 42, no. 12 (1998): 3218-3224), metastasis of cancerous cells (McCawley, L. J. and L. M. Matrisian Current Opinion in Cell Biology, 13 (2001): 534-540), tissue damage in periodontal disease (Sandholm, L. Journal of Clinical Periodontology, 13, no. 1 (1986): 19-26), complications in pregnancy (Locksmith, G. J. et al. Am J Obstet Gynecol, 184, no. 2 (January 2001): 159-164), tissue destruction in inflamed joints (Cunnane, G. et al. Arthritis & Rheumatism, 44, no. 8 (2001): 1744-1753), and destruction of pro-healing factors and nascent tissue in chronic, non-healing, wounds (Ladwig, G. P. et al. Wound Repair and Regeneration, 10 (2002): 26-37; Trengove, N. J. et al. Wound Repair and Regeneration, 7 (1999): 442-452; Yager, D. R. et al. Wound Repair and Regeneration, 5 (1997): 23-32).
Studies of proteases in diseases have employed tests from one of two (or a combination of the two) classes: molecular presence-based tests, or catalytic activity-based tests. A common molecular presence-based test would be an immuno-detection assay where the protease of interest is isolated from the rest of the sample and antibodies that specifically recognize that protease are labeled with a detectable agent. The other class, catalytic activity-based, does not just measure whether the molecule (or the portion of the molecule that an antibody recognizes) is present, it measures how active the molecule is in the given conditions.
Currently, three protease activity based assays are in common laboratory use: the zymogram (Quesada, A. R. et al. Clin. Exp. Metastasis, 15 (1997): 26-32), the thiopeptolide continuous colorimetric assay (Stein, R. L. and M. Izquierdo-Martin Archives of Biochemistry and Biophysics, 308, no. 1 (January 1994): 274-277; Oxford Biomedical Research. Colorimetric Drug Discovery Assay for Matrix Metalloproteinase-7, Product Brochure, Oxford, Mich.: Oxford Biomedical Research, 2005 Oxford Biomedical Research. Colorimetric Drug Discovery Assay for Matrix Metalloproteinase-7, Product Brochure, Oxford, Mich.: Oxford Biomedical Research, 2005; Rosa-Bauza, Y. T. et al. ChemBioChem, 8 (2007): 981-984), and the fluorescence resonance energy transfer (FRET) continuous fluorometric assay (Fairclough, R. H. and C. R. Cantor Methods in Enzymology, 48 (1978): 347-379; Stryer, L. Annu Rev Biochem, 47 (1978): 819-846; Yaron, A. et al. Analytical Biochemistry, 95, no. 1 (May 1979): 228-235; Matayoshi, E. D. et al. Science, 247 (February 1990): 954-958; Beekman, B. et al. FEBS Letters, 390, no. 2 (1996): 221-225; Knäuper, V. et al. The Journal of Biological Chemistry, 271, no. 3 (January 1996): 1544-1550).
The zymogram is usually used when analyzing mixtures of proteases since it first resolves the different proteases by mass and then measures their activity. The thiopeptolide assay is used by suppliers of proteases to verify/guarantee a basic level of protease activity in the supplied sample (Calbiochem Data Sheet PF024 Rev. 25-September-06 RFH) (Biomol Product Data Catalog No.: SE-244).
Many currently marketed rapid, point-of-care diagnostic technologies are limited by their analytical sensitivity or by the number of analytes detected in a single assay.